The renal epithelial sodium channel (ENaC) plays a very important role in maintaining Na + homeostasis, and consequently controls systemic blood pressure. Although the regulation of ENaC has been extensively studied during the past decade, the regulation of ENaC by membrane phospholipids remains largely unexplored. Preliminary data in this application have shown that ENaC is regulated by the membrane concentration of anionic phospholipids such as phosphatidylinosotol 4,5-bisphosphate (PIP2) and phosphatidylinosotol 3,4,5- trisphosphate (PIP3). In addition, physiologically, ATP binding to purinergic P2Y receptors appears to be a primary regulator of anionic phospholipids and, thereby, a regulator of ENaC. ATP inhibits ENaC by P2v receptor activation of phospholipase C (PLC) that degrades PIPe, and the reduction in PIP2 reduces ENaC activity. Besides P2Y receptors, another physiological regulator of ENaC, the steroid hormone aldosterone, also alters the amount of anionic phospholipids. Therefore, I hypothesize that PIP2 and PIP3 might mediate the regulation of ENaC by ATP and aldosterone. The first specific aim of this proposal will be focused on determining whether ATP inhibits ENaC by decreasing PIP2 concentration and whether aldosterone stimulates ENaC by increasing PIP3 concentration. Patch-clamp studies of ENaC activity will be correlated to laser confocal microscopy localization of membrane PIP2 and PIP3 with specific GFP-fused pleckstrin homology domains. Since the cytoplasmic domains of beta- and gamma-ENaCs contain lysine- and arginine-rich motifs, I hypothesize that these motifs might represent selective PIP2 and PIP3 binding sites. The second specific aim will be focused on determining whether truncations and point mutations of the putative PIP2 and PIP3 binding sites abolish both the binding of ENaC to anionic phospholipids and the effects of ATP and aldosterone on ENaC activity and cell surface expression. Electrophysiological studies will be correlated to both lipid-protein overlay assays (immunoprecipitation) and laser confocal microscopy analysis of anionic phospholipids levels and ENaC cell surface expression. These studies will provide direct evidence for further understanding the downstream pathways for receptor-mediated regulation of ENaC.